鉴定坏死分枝杆菌白细胞毒素蛋白上的线性 B 细胞表位。

IF 2.5 3区生物学 Q3 MICROBIOLOGY

Anaerobe Pub Date : 2024-07-25 DOI:10.1016/j.anaerobe.2024.102884

Jiawei Xiao , Siwen Yu , Kai Jiang , Xianjing He , Lan Bi , Pengyu Zhao , Tianshuo Wang , Ning Yang , Donghua Guo

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引用次数: 0

摘要

目的：坏死杆菌可导致人类患上莱米埃尔综合征，动物患上一系列疾病，包括烂脚病和肝脓肿。坏死杆菌释放的主要毒力因子是白细胞毒素，已被证明与疾病的严重程度密切相关。白细胞毒素通常被用作亚单位疫苗的关键抗原。因此，有必要鉴定 F. necrophorum 白细胞毒素的 B 细胞表位：在这项研究中，我们利用淋巴细胞杂交瘤技术开发出了一种针对F. necrophorum白细胞毒素蛋白的单克隆抗体（mAb）3D7。利用白细胞毒素截短重组蛋白和肽，通过Western印迹、ELISA和点印迹，并通过SWISS-MODEL同源建模和PyMOL可视化，鉴定了3D7 mAb识别的B细胞表位：结果：经鉴定，3D7 mAb 属于具有 κ 链轻链的 IgG1 亚类。它与天然白细胞毒素具有反应性。结果表明，3D7 mAb 能识别 F. necrophorum 白细胞毒素蛋白的 B 细胞表位 I2168SSFGVGV2175（EP-3D7）。序列对比分析表明，EP-3D7 在 F. necrophorum 菌株中高度保守，但在其他细菌中保守程度较低，这表明了 EP-3D7 的特异性。EP-3D7以β折叠的方式存在于白细胞毒素蛋白的表面：总之，这些结果确定了 EP-3D7 是 F. necrophorum 白细胞毒素的保守抗原表位。结论：这些结果确立了 EP-3D7 作为 F. necrophorum 白细胞毒素的保守抗原表位，它对疫苗和 F. necrophorum 表位诊断试剂的开发可能很有价值。

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Identification of linear B cell epitopes on the leukotoxin protein of Fusobacterium necrophorum

Objective

Fusobacterium necrophorum can casuse Lemierre's syndrome in humans and a range of illnesses, including foot rot and liver abscesses, in animals. The main virulence factor released by F. necrophorum is leukotoxin, which has been shown to have a strong correlation with the severity of the disease. Leukotoxin is commonly employed as the key antigen in the formulation of subunit vaccines. Therefore, identification of the B-cell epitope of F. necrophorum leukotoxin is necessary.

Methods

In this research, we utilized lymphocyte hybridoma technology to develop a monoclonal antibody (mAb), 3D7, targeting the F. necrophorum leukotoxin protein. Identification of B-cell epitopes recognized by 3D7 mAb was achieved through Western blot, ELISA and dot blots using leukotoxin-truncated recombinant proteins and peptides, and through SWISS-MODEL homology modeling and PyMOL visualization.

Results

The 3D7 mAb was identified as belonging to the IgG1 subclass with a κ-chain light chain. It demonstrated reactivity with the natural leukotoxin. The results showed that the 3D7 mAb recognizes a B-cell epitope of the F. necrophorum leukotoxin protein, I²¹⁶⁸SSFGVGV²¹⁷⁵ (EP-3D7). Sequence comparison analysis showed that EP-3D7 was highly conserved in F. necrophorum strains, but less conserved in other bacteria, indicating the specificity of EP-3D7. EP-3D7 is present on the surface of leukotoxin proteins in a β-folded manner.

Conclusions

In summary, these results establish EP-3D7 as a conserved antigenic epitope of F. necrophorum leukotoxin. It could be valuable in the development of vaccines and diagnostic reagents for F. necrophorum epitopes.

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来源期刊

Anaerobe 生物-微生物学

CiteScore

5.20

自引率

8.70%

发文量

137

审稿时长

76 days

期刊介绍： Anaerobe is essential reading for those who wish to remain at the forefront of discoveries relating to life processes of strictly anaerobes. The journal is multi-disciplinary, and provides a unique forum for those investigating anaerobic organisms that cause infections in humans and animals, as well as anaerobes that play roles in microbiomes or environmental processes. Anaerobe publishes reviews, mini reviews, original research articles, notes and case reports. Relevant topics fall into the broad categories of anaerobes in human and animal diseases, anaerobes in the microbiome, anaerobes in the environment, diagnosis of anaerobes in clinical microbiology laboratories, molecular biology, genetics, pathogenesis, toxins and antibiotic susceptibility of anaerobic bacteria.